We have recently discovered that calmodulin is a major component of trichocysts, rod-like structures released by Ca-dependent exocytosis from the ciliate protozoan Paramecium. More than 95% of the protein of this organelle is calmodulin and calmodulin-like proteins. This remarkably high calmodulin content suggests that in addition to its well-known role as regulator of cellular processes, calmodulin plays a structural role in this simple eucaryote. Trichocysts may, therefore, provide a unique opportunity for studies of the structure and function of this ubiquitous Ca-binding protein. We have also purified to homogeneity a Ca-dependent ATPase which is released from Paramecium under conditions which cause trichocyst release, and we have found that antibodies against this ATPase specifically interact with trichocysts. I propose to study the role of calmodulin, calmodulin-like proteins, and this Ca-ATPase in the structure and assembly of Paramecium trichocysts. Unreleased secretory vesicles containing the precursors of trichocysts will be isolated, and their content of calmodulin, calmodulin-like proteins and Ca-ATPase will be compared with that of the secreted product. The assembly of trichocysts from these secretory precursors will be studied in vitro, and the effects of Ca++, Ca++ antagonists, calmodulin antagonists, purified Ca-ATPase, and antibody against calmodulin and against Ca-ATPase upon the in vitro assembly will be determined. Three-dimensional image reconstructions from electron micrographic images will be attempted. Two morphologically distinct regions of the extruded trichocyst (tip and shaft) will be physically separated, and the distribution of calmodulin and of Ca-ATPase between them will be studied. The relationship between calmodulin and other calmodulin-like proteins of trichocysts will be studied. Their affinity for Ca++ and for calmodulin antagonists will be determined; their immunological cross-reactivity will be tested; their trypticand cyanogen bromide peptide maps will be compared; and the patterns of N-methylation and carboxymethylation will be determined for each.